The purpose of this study is to determine the relationship between cardiac performance and energy production in isolated rat heart when heart function is modified either by calcium concentration or by oxygen partial pressure (PO2), and to evaluate the relative contribution of glycolytic ATP. Hearts are perfused at a constant 10 ml/min flow and submitted to increasing calcium concentration (0.36 to 1.78 mM free calcium) with maximal PO2 or to graded hypoxia (660 to 52 mmHg) with maximal calcium concentration. Cardiac performance, oxygen consumption (VO2), lactate+pyruvate production are measured. To inhibit glycolysis, perfusions are also carried out with deoxyglucose (2-DG). The plotting of mitochondrial ATP production, as calculated from VO2 vs contractility parameters shows a different relationship when we modify the PO2 or the calcium concentration, whereas the relationship is similar for heart rate. When cardiac performance is related to total ATP production, glycolytic ATP being calculated from lactate+pyruvate production, the difference, although decreased, remains. 2-DG impairs heart function, but with 2-DG the relationship between ATP production and heart function becomes unique. In conclusion, there is an evident difference in the dependence of heart contractility on ATP production according to the factor that limits heart function. The contribution of glycolysis to energy production does not explain all of this difference. Furthermore, such a difference does not exist for heart rate. This raises the question of energy compartmentation in myocardial cells.